Transcriptome differences in the hypopharyngeal gland between Western Honeybees (Apis mellifera) and Eastern Honeybees (Apis cerana)

Background

Apis mellifera and Apis cerana are two sibling species of Apidae. Apis cerana is adept at collecting sporadic nectar in mountain and forest region and exhibits stiffer hardiness and acarid resistance as a result of natural selection, whereas Apis mellifera has the advantage of producing royal jelly. To identify differentially expressed genes (DEGs) that affect the development of hypopharyngeal gland (HG) and/or the secretion of royal jelly between these two honeybee species, we performed a digital gene expression (DGE) analysis of the HGs of these two species at three developmental stages (newly emerged worker, nurse and forager).

Results

Twelve DGE-tag libraries were constructed and sequenced using the total RNA extracted from the HGs of newly emerged workers, nurses, and foragers of Apis mellifera and Apis cerana. Finally, a total of 1482 genes in Apis mellifera and 1313 in Apis cerana were found to exhibit an expression difference among the three developmental stages. A total of 1417 DEGs were identified between these two species. Of these, 623, 1072, and 462 genes showed an expression difference at the newly emerged worker, nurse, and forager stages, respectively. The nurse stage exhibited the highest number of DEGs between these two species and most of these were found to be up-regulated in Apis mellifera. These results suggest that the higher yield of royal jelly in Apis mellifera may be due to the higher expression level of these DEGs.

Conclusions

In this study, we investigated the DEGs between the HGs of two sibling honeybee species (Apis mellifera and Apis cerana). Our results indicated that the gene expression difference was associated with the difference in the royal jelly yield between these two species. These results provide an important clue for clarifying the mechanisms underlying hypopharyngeal gland development and the production of royal jelly.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-744) contains supplementary material, which is available to authorized users.     

Uncovering the novel characteristics of Asian honey bee,Apis cerana,by whole genome sequencing

Background

The honey bee is an important model system for increasing understanding of molecular and neural mechanisms underlying social behaviors relevant to the agricultural industry and basic science. The western honey bee, Apis mellifera, has served as a model species, and its genome sequence has been published. In contrast, the genome of the Asian honey bee, Apis cerana, has not yet been sequenced. A. cerana has been raised in Asian countries for thousands of years and has brought considerable economic benefits to the apicultural industry. A cerana has divergent biological traits compared to A. mellifera and it has played a key role in maintaining biodiversity in eastern and southern Asia. Here we report the first whole genome sequence of A. cerana.

Results

Using de novo assembly methods, we produced a 238 Mbp draft of the A. cerana genome and generated 10,651 genes. A.cerana-specific genes were analyzed to better understand the novel characteristics of this honey bee species. Seventy-two percent of the A. cerana-specific genes had more than one GO term, and 1,696 enzymes were categorized into 125 pathways. Genes involved in chemoreception and immunity were carefully identified and compared to those from other sequenced insect models. These included 10 gustatory receptors, 119 odorant receptors, 10 ionotropic receptors, and 160 immune-related genes.

Conclusions

This first report of the whole genome sequence of A. cerana provides resources for comparative sociogenomics, especially in the field of social insect communication. These important tools will contribute to a better understanding of the complex behaviors and natural biology of the Asian honey bee and to anticipate its future evolutionary trajectory.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-16-1) contains supplementary material, which is available to authorized users.     

幼虫信息素中三种酯类对中华蜜蜂工蜂发育和采集行为的影响

为研究中华蜜蜂Apis cerana cerana幼虫信息素中的3种酯类成分(甲基棕榈酸酯、乙基棕榈酸酯和乙基油酸酯)对其工蜂发育和采集行为的影响, 取1日龄工蜂喂食不同食物, 把喂食食物(炼糖)中添加0.1%或1%(w/w)某种酯类的工蜂组作为处理组, 以喂食食物(炼糖)中不添加酯类(0%)的工蜂组作为对照组, 然后测定7日龄、14日龄工蜂的卵巢发育和3, 5, 7, 12, 18和21日龄工蜂王浆腺的宽度, 以及工蜂首次参加采集的日龄。结果表明: (1)在无王群中, 甲基棕榈酸酯两处理组(1%, 0.1%)的7日龄和14日龄工蜂卵巢发育率都显著低于对照组; 在无王群中, 0.1%乙基棕榈酸酯处理组的14日龄工蜂卵巢发育率极显著低于对照组; 在有王群中, 1%甲基棕榈酸酯处理组和乙基油酸酯两处理组的14日龄工蜂卵巢发育都显著低于对照组;(2)在有王群中, 甲基棕榈酸酯两处理组的5日龄和7日龄工蜂王浆腺宽度显著大于对照组, 12, 18, 21日龄工蜂王浆腺宽度极显著小于对照组; 乙基油酸酯两处理组的7~21日龄工蜂王浆腺宽度显著小于对照组;(3)3种酯类, 只有甲基棕榈酸酯处理组的工蜂首次采集日龄显著大于对照组。这些结果说明, 不同蜜蜂幼虫信息素对中华蜜蜂工蜂发育和采集行为具有不同的生物学效应。     

中华蜜蜂工蜂视叶胚后发育过程中的细胞凋亡

本研究通过形态解剖和原位末端标记法(TUNEL),对中华蜜蜂Apis cerana cerana视叶胚后发育过程中的细胞凋亡进行了研究,结果表明:视叶内的细胞程序性死亡开始出现在1龄幼虫末期,随后凋亡细胞数量逐渐增加;在视叶的胚后发育过程中,细胞凋亡经历了3个高峰期,即2龄幼虫、5龄幼虫和蛹发育的第2天;在视叶3个部分的发育中,视髓层中细胞凋亡的数量远远多于视小叶和视神经节层,而视神经节层最少,说明了细胞凋亡的数量和位置与各部分结构发育的时间以及神经投射有关。广泛的细胞凋亡是蜜蜂视叶发育过程中的一个显著特征。     

中华蜜蜂工蜂咽下腺胚后发育过程中细胞的增殖和凋亡

【目的】咽下腺(hypopharyngeal gland)是蜜蜂重要的外分泌腺,是工蜂合成和分泌蜂王浆的主要腺体。本研究的目的在于了解中华蜜蜂Apis cerana cerana工蜂咽下腺的胚后发育特点。【方法】通过组织形态学、Brd U免疫组织化学和TUNEL细胞凋亡检测等技术,对中华蜜蜂工蜂咽下腺的胚后发育过程及组织结构特点进行了比较研究。【结果】中华蜜蜂工蜂的咽下腺起源自预蛹阶段口器内壁的陷入,细胞分裂活动的高峰期集中在蛹发育的前3 d,随后分裂细胞数减少,并一直持续到蛹发育的第7天左右结束;分泌腺泡的出现大约在蛹发育的第5天。到蛹发育的末期,咽下腺已基本形成,但是没有发育完全;哺育蜂的咽下腺高度发育,分泌活动旺盛;采集蜂的咽下腺中有许多分泌细胞凋亡。【结论】本研究揭示了中蜂工蜂咽下腺胚后发育过程中细胞增殖和凋亡的模式,为昆虫咽下腺的发育和功能研究提供了一定的理论依据。     

中华蜜蜂工蜂cDNA文库的构建及ESTs测序分析

【目的】为了解中华蜜蜂Apis cerana cerana工蜂的抗逆性,构建了中华蜜蜂工蜂的cDNA文库,并对文库质量进行分析。【方法】本研究利用SMART技术构建了中华蜜蜂工蜂的全长cDNA文库。【结果】文库库容为3.6×106 cfu/m L,文库重组率为97%,插入片段长度多数分布在1 000 bp左右。挑取cDNA克隆进行EST测序,共进行了306个成功反应,软件拼接共得到234个单基因簇(Unigene),其中包括207个单拷贝(Singletons)序列及27个重叠群(Contigs)。使用Blastx将这些序列同Gen Bank等数据库进行查询、比对和注释,结果显示141条序列有相关同源性,其他序列没有明显的同源性,这也为我们发现新功能基因提供了可靠依据。【结论】此文库的构建在中华蜜蜂功能基因的分离、克隆、筛选以及基因功能研究等方面具有重要作用。     

Occurrence and prevalence of seven bee viruses in Apis mellifera and Apis cerana apiaries in China

Populations of Apis mellifera and Apis cerana in China were surveyed for seven bee viruses: acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV), sacbrood virus (SBV), and Isreal acute paralysis virus (IAPV). No KBV was detected from any samples of the two species. In A. mellifera, DWV was the most prevalent virus, but in A. cerana, SBV was the dominant. Simultaneous multiple infections of viruses were common in both species. This is the first report of detection of IAPV and CBPV in A. cerana.     

Prevalence and distribution of six bee viruses in Korean Apis cerana populations

The prevalence and distribution of six bee viruses was investigated in 527 Apis cerana samples which were collected from five provinces in South Korea. The most prevalent virus, black queen cell virus (BQCV), was present in 75.11% of 446 adult bee samples, followed by sacbrood virus (SBV) in 30.71%. Deformed wing virus (DWV), Kashmir bee virus (KBV), and chronic bee paralysis virus (CBPV) were present at lower levels of 8.07%, 1.56%, and 0.44%, respectively. The most prevalent virus in 81 larvae samples was SBV, with an incidence of 60.49%, followed by BQCV in 48.14%, DWV in 6.17%, and KBV in 1.23% of samples. CBPV infection was not detected in larvae samples, and acute bee paralysis virus (ABPV) was not present in both larvae and adult bee. Simultaneous infections with up to four viruses were also identified. Of these, infections with SBV and BQCV were most frequent in 25.61% of samples. The distribution of these viruses varied considerably throughout the geographic regions investigated. The three provinces of Gyeongbuk, Jeonnam, and Chungbuk had the highest frequency of bee viruses.     

The relationship between population size,amount of brood,and individual foraging behaviour in the honey bee,Apis mellifera L.

This study experimentally examines the relationship between colony state and the behaviour of individual pollen and nectar foragers in the honey bee, Apis mellifera L. In the first experiment we test the prediction that individual pollen foragers from colonies with higher brood quantities should exhibit a greater work effort for pollen resources than individual pollen foragers from colonies with low brood quantities. Eight colonies were assigned into two treatment groups; HIGH brood colonies were manipulated to contain 9600±480 cm² brood area; LOW brood colonies were manipulated to contain 1600±80 cm² brood area. We measured colony brood levels over the course of the experiment and collected individual pollen loads from returning pollen foragers. We found that, while colonies remained significantly different in brood levels, individual pollen foragers from HIGH brood colonies collected larger loads than individuals from LOW brood colonies. In the second experiment we investigated the influence of colony size on the behaviour of individual nectar foragers. We assigned eight colonies to two treatment groups; LARGE colonies were manipulated to contain 35000±1700 adult workers with 3500±175 cm² brood area, and SMALL colonies were manipulated to contain 10000±500 adult workers with 1000±50 cm² brood area. We observed foraging trips of individually marked workers and found that individuals from LARGE colonies made longer foraging trips than those from SMALL colonies (LARGE: 1666.7±126.4 seconds, SMALL: 1210.8±157.6 seconds), and collected larter nectar loads (LARGE: 19.2±1.0 l, SMALL: 14.6±0.8 l). These results indicate that individual nectar foragers from LARGE colonies tend to work harder than individuals from SMALL colonies. Both experiments indicate that the values of nectar and pollen resources to a colony change depend on colony state, and that individual foragers modify their behaviour accordingly.     

An ecological digest of the small hive beetle (Aethina tumida), a symbiont in honey bee colonies (Apis mellifera)

The small hive beetle (Aethina tumida Murray) is an endemic scavenger in colonies of western honey bee subspecies (Apis mellifera L.) inhabiting sub-Saharan Africa where it only occasionally damages host colonies. Such damage is usually restricted to weakened/diseased colonies or is associated with after absconding events (all bees, including the queen, leave the hive) due to behavioral resistance mechanisms of its host. In sharp contrast, the beetle has proven deleterious to honey bee colonies in introduced ranges of the United States and Australia. With this review we synthesize the existing data in a manner that allows us to discuss the beetle’s natural history from an ecological perspective. A thorough exploration of beetle ecology allows us to 1) illuminate the unique symbiotic relationship it and its host share and understand how this relationship is fostered, 2) place this relationship in context with those of other arthropods inhabiting social insect colonies, 3) understand its natural reliance on honey bee colonies, 4) predict its spread outside its native range, and 5) predict its effects on non-African honey bees and non-target species. Here we present an amalgamation of information that will contribute to a more thorough and appropriate understanding of not only small hive beetles as symbionts, but of social insect symbionts in general. Received 4 April 2005; revised 15 October 2005; accepted 18 October 2005.     

贵州越夏期与越冬期中华蜜蜂病毒病发生的调查

蜜蜂是重要的传粉昆虫,在农业生产和生态平衡中起着重大作用。近年来蜜蜂数量大幅下降,由于农药使用、生态污染、气候变化等原因,以及受到天敌如胡蜂Vespidae的影响,更重要的是受到病原如真菌、细菌以及各种病毒的危害。其中蜜蜂病毒病是造成蜜蜂数量减少的重要原因之一。为了调查贵州省越夏期和越冬期中华蜜蜂感染病毒病的情况,利用RT-PCR技术对贵州省兴义市、息烽县、台江县、龙里县采集的样本进行检测。结果显示：贵州省越夏期以红火蚁Solenopsis invicta Buren病毒Sindbis virus（SINV）为主要流行病毒,而越冬期以囊状幼虫病毒Sacbrood virus（SBV）和黑蜂王台病毒Black queen cell virus（BQCV）为主要流行病毒。本研究初步调查了贵州省主要中蜂饲养区在两个重要饲养阶段发生蜜蜂病毒病情况,这将在一定程度上为防治蜜蜂病毒病提供理论依据。     

Social parasitism by workers in queenless and queenright Apis cerana colonies

We examined worker reproduction in queenless and queenright Apis cerana colonies to determine if they are parasitized by workers from other nests. The results demonstrate that 2-6% of workers in queenright colonies are from another nest (non-natal), but these workers are not statistically more likely to have activated ovaries than natal workers, and are therefore unlikely to be active parasites. However, in queenless colonies we found a significant difference between the proportion of non-natal (72.7%) and natal (36.3%) workers with activated ovaries. Non-natal workers also had significantly higher reproductive success than natal workers: 1.8% of workers were non-natal, but these laid 5.2% of the eggs and produced 5.5% of the pupae. Unlike A. florea, the proportion of non-natal workers does not increase in queenless nests.     

The interplay between dancing and trophallactic behavior in the honey bee Apis mellifera

The interplay between the recruitment dance and food-giving trophallactic contacts of returning Apis mellifera foragers was analyzed. Dancing and trophallactic events were recorded for bees returning from a rate feeder that provided 50% weight on weight sucrose solution at a constant flow rate of 5 μl min⁻¹. Bees that had danced immediately before their trophallactic contact had more recipients per trophallaxis compared with bees that did not dance before. Thus, besides information coded in dancing behavior, dance maneuvers could serve as a stimulus to increase attention of bees located on the dance floor to receive nectar. In addition, the number of bees receiving food during a trophallaxis showed a positive correlation with the probability of dancing immediately after contacting. The time from arrival at the hive to when the first or the subsequent contacts took place presented no correlation with the probability of dancing after trophallaxis. Also, the duration of a trophallaxis was positively correlated with the number of recipients per trophallaxis. These results suggest that returning foragers could receive information during a trophallactic contact with their hive mates that modify thresholds for dancing. Dance maneuvers and trophallactic contacts performed by foraging bees seem to be “mutually” affected. Accepted: 29 November 1999     

Genetic reincarnation of workers as queens in the Eastern honeybee Apis cerana

Thelytokous parthenogenesis, or the asexual production of female offspring, is rare in the animal kingdom, but relatively common in social Hymenoptera. However, in honeybees, it is only known to be ubiquitous in one subspecies of Apis mellifera, the Cape honeybee, A. mellifera capensis. Here we report the appearance of queen cells in two colonies of the Eastern honeybee Apis cerana that no longer contained a queen or queen-produced brood to rear queens from. A combination of microsatellite genotyping and the timing of the appearance of these individuals excluded the possibility that they had been laid by the original queen. Based on the genotypes of these individuals, thelytokous production by natal workers is the most parsimonious explanation for their existence. Thus, we present the first example of thelytoky in a honeybee outside A. mellifera. We discuss the evolutionary and ecological consequences of thelytoky in A. cerana, in particular the role thelytoky may play in the recent invasions by populations of this species.     

Laying workers in queenless honeybee (Apis mellifera L.) colonies have physiological states similar to that of nurse bees but opposite that of foragers

Honeybee workers shift their labors from nursing their brood to foraging according to their age after eclosion. When the queen is lost from the colony, however, some workers become 'laying workers' whose ovaries develop to lay eggs. Here we investigated whether the physiological state of laying workers is more similar to that of nurse bees or foragers by examining the hypopharyngeal gland (HPG) and hemolymph vitellogenin titers. In a normal colony, nurse bees have well-developed HPGs that synthesize 'major royal jelly proteins' and high hemolymph vitellogenin titers, whereas foragers have shrunken HPGs that synthesize 70-kDa alpha-glucosidase and low hemolymph vitellogenin titers. In queenless colonies, however, workers with developed ovaries (laying workers) tended to have more developed HPGs and to synthesize major royal jelly proteins, whereas workers with shrunken HPGs tended to synthesize alpha-glucosidase and to have undeveloped ovaries. Furthermore, the workers with developed ovaries had higher vitellogenin titers than nurse bees, whereas those with undeveloped ovaries had lower vitellogenin titers. These findings indicate that the physiological state of laying workers is similar to that of nurse bees, but opposite that of foragers.     

Effects of sequential exposures of sub-lethal doses of amitraz and thiacloprid on learning and memory of honey bee foragers,Apis mellifera

Pollinators, honey bees in particular, are continuously exposed to various mixtures of pesticides, which contribute to their population decline. Both amitraz and thiacloprid have been proven less toxic to honey bees and are frequently applied in- and out-hive, respectively. We examined the sub-lethal effects of amitraz, thiacloprid and their sequential exposure on learning, memory and sugar responsiveness in Apis mellifera using the Proboscis extension response (PER). Sub-lethal doses of amitraz (0.1, 0.2 and 0.4 µg/bee) and thiacloprid (0.05, 0.1 and 0.2 µg/bee) were tested. Sub-lethal effects were observed only at the highest doses of each pesticide treatment; amitraz (0.4 µg/bee) and thiacloprid (0.2 µg/bee) but not in lower doses. In sequential treatment of amitraz and thiacloprid, reduced acquisition and memory retention were significant across all tested doses. The same profile was also obtained on sugar responsiveness of foragers. Our results suggest that the sequential exposure would pose higher risk to honey bee compared to single pesticide exposure by reducing the bees’ appetitive olfactory learning, memory and sugar acuity more than individual pesticide exposures.